The terminology of microbial genetics is sufficiently complicated that certain definitions will be particularly useful in the understanding of this invention:
Extrachromosomal element. . . a hereditary unit that is physically separate from the chromosome of the cell; the terms "extrachromosomal element" and "plasmid" are synonymous; when physically separated from the chromosome, some plasmids can be transmitted at high frequency to other cells, the transfer being without associated chromosomal transfer;
Episome . . . a class of plasmids that can exist in a state of integration into the chromosome of their host cell or as an autonomous, independently replicating, cytoplasmic inclusion;
Transmisible plasmid . . . a plasmid that carries genetic determinants for its own intercell transfer via conjugation;
DNA . . . deoxytribonucleic acid;
Bacteriophage . . . a particle composed of a piece of DNA encoded and contained within a protein head portion and having a tail and tail fibers composed of protein;
Transducing phage . . . a bacteriophage that carries fragments of bacterial chromosomal DNA and transfers this DNA on subsequent infection of another bacterium;
Conjugation . . . the process by which a bacterium establishes cellular contact with another bacterium and the transfer of genetic material occurs;
Curing . . . the process by which selective plasmids can be eliminated from the microorganism;
Curing agent . . . a chemical material or a physical treatment that enhances curing;
Genome . . . a combination of genes in some given sequence;
Degradative pathway . . . a sequence of enzymatic reactions (e.g. 5 to 10 enzymes are produced by the microbe) converting the primary substrate to some simple common metabolite, a normal food substance for microorganisms;
(Sole carbon source).sup.-. . . indicative of a mutant incapable of growing on the given sole carbon source;
(Plasmid).sup.del . . . indicative of cells from which the given plasmid has been completely driven out by curing or in which no portion of the plasmid ever existed;
(Plasmid).sup.-. . . indicative of cells lacking in the given plasmid; or cells harboring a non-functional derivative of the given plasmid;
(Amino-acid).sup.-. . . indicative of a strain that cannot manufacture the given amino acid;
(Vitamin).sup.-. . . indicative of a strain that cannot manufacture the given vitamin and
(Plasmid).sup.+. . . indicates that the cells contain the given plasmid.
Plasmids are believed to consist of double-stranded DNA molecules. The genetic organization of a plasmid is believed to include at least one replication site and a maintenance site for attachment thereof to a structural component of the host cell. Generally, plasmids are not essential for cell viability.
Much work has been done supporting the existence, functions and genetic organization of plasmids. As is reported in the review by Richard P. Novick "Extrachromosomal Inheritance in Bacteria" (Bacteriological Reviews, June 1969, pp. 210-263, [1969]) on page 229, "DNA corresponding to a number of different plasmids has been isolated by various methods from plasmid-positive cells, characterized physiochemically and in some cases examined in the electron microscope".
There is no recognition in the Novick review of the existence of energy-generating plasmids specifying degradative pathways. As reported on page 237 of the Novick review, of the known (non energy-generating) plasmids "Combinations of four or five different plasmids in a cell seem to be stable."
Plasmids may be compatible (i.e. they can reside stably in the same host cell) or incompatible (i.e. they are unable to reside stably in a single cell). Among the known plasmids, for example, are sex factor plasmids and drug-resistance plasmids.
Also, as stated on page 240 of the Novick review, "Cells provide specific maintenance systems or sites for plasmids. It is though that attachment of such sites is required for replication and for segregation of replicas. Each plasmid is matched to a particular maintenance site . . . ". Once a plasmid enters a given cell, if there is no maintenance site available, because of prior occupancy by another plasmid, these plasmids will be incompatible.
The biodegradation of aromatic hydrocarbons such as phenol, cresols and salicylate has been studied rather extensively with emphasis on the biochemistry of these processes, notably enzyme characterization, nature of intermediates involved and the regulatory aspects of the enzymic actions. The genetic basis of such biodegradation, on the other hand, has not been as thoroughly studied because of the lack of suitable transducing phages and other genetic tools.
The work of Chakrabarty and Gunsalus (Genetics, 68, No. 1, page S10, [1971]) has showed that the genes governing the synthesis of the enzymes responsible for the degradation of camphor constitute a plasmid. Similarly, this work has shown the plasmid nature of the octane-degradative pathway. However, attempts by the authors to provide a microorganism with both CAM and OCT plasmids were unsuccessful, these plasmids being incompatible.
In Escherichia coli artificial, transmissible plasmids (one per cell) have been made, each containing a degradative pathway. These plasmids, not naturally occurring, are F'lac and F'gal, wherein the lactose-and galactose-degrading genes were derived from the chromosome of the organism. Such plasmids are described in "F-prime Factor Formation in E. Coli K12" by J. Scaife (Genet. Res. Cambr. [1966], 8, pp. 189-196).
If the development of microorganisms containing multiple containing energy-generating plasmids specifying preselected degradative pathways could be made possible, the economic and environmental impact of such an invention would be vast. For example, there would be immediate application for such versatile microbes in the production of proteins from hydrocarbons ("Proteins from Petroleum"--Wang, Chemical Engineering, August 26, 1968, page 99); in cleaning up oil spills ("Oil Spills: An Environmental Threat"--Environmental Sciene and Technology, Volume 4, February 1970, page 97); and in the disposal of used automotive lubricating oils ("Waste Lube Oils Pose Disposal Dilemma", Environmental Science and Technology, Volume 6, page 25, January 1972).